Compounds and compositions for delivering active agents

ABSTRACT

Compounds and compositions for the delivery of active agents are provided. Methods of administration and preparation are provided as well.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/272,726, filed Mar. 1, 2001; U.S. ProvisionalApplication No. 60/314,783, filed Aug. 24, 2001; and U.S. ProvisionalApplication No. 60/323,139, filed Sep. 17, 2001, all of which are herebyincorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to compounds for delivering activeagents, such as biologically or chemically active agents, to a target.These compounds are well suited for forming non-covalent mixtures withactive agents for oral, intracolonic, pulmonary, and other routes ofadministration to animals. Methods for the preparation andadministration of such compositions are also disclosed.

BACKGROUND OF THE INVENTION

[0003] Conventional means for delivering active agents are oftenseverely limited by biological, chemical, and physical barriers.Typically, these barriers are imposed by the environment through whichdelivery occurs, the environment of the target for delivery, and/or thetarget itself. Biologically and chemically active agents areparticularly vulnerable to such barriers.

[0004] In the delivery to animals of biologically active and chemicallyactive pharmacological and therapeutic agents, barriers are imposed bythe body. Examples of physical barriers are the skin, lipid bi-layersand various organ membranes that are relatively impermeable to certainactive agents but must be traversed before reaching a target, such asthe circulatory system. Chemical barriers include, but are not limitedto, pH variations in the gastrointestinal (GI) tract and degradingenzymes.

[0005] These barriers are of particular significance in the design oforal delivery systems. Oral delivery of many biologically or chemicallyactive agents would be the route of choice for administration to animalsif not for biological, chemical, and physical barriers. Among thenumerous agents which are not typically amenable to oral administrationare biologically or chemically active peptides, such as calcitonin andinsulin; polysaccharides, and in particular mucopolysaccharidesincluding, but not limited to, heparin; heparinoids; antibiotics; andother organic substances. These agents may be rapidly renderedineffective or destroyed in the gastro-intestinal tract by acidhydrolysis, enzymes, and the like. In addition, the size and structureof macromolecular drugs may prohibit absorption.

[0006] Earlier methods for orally administering vulnerablepharmacological agents have relied on the co-administration of adjuvants(e.g., resorcinols and non-ionic surfactants such as polyoxyethyleneoleyl ether and n-hexadecylpolyethylene ether) to increase artificiallythe permeability of the intestinal walls, as well as theco-administration of enzymatic inhibitors (e.g., pancreatic trypsininhibitors, diisopropylfluorophosphate (DFF) and trasylol) to inhibitenzymatic degradation. Liposomes have also been described as drugdelivery systems for insulin and heparin. However, broad spectrum use ofsuch drug delivery systems is precluded because: (1) the systems requiretoxic amounts of adjuvants or inhibitors; (2) suitable low molecularweight cargos, i.e. active agents, are not available; (3) the systemsexhibit poor stability and inadequate shelf life; (4) the systems aredifficult to manufacture; (5) the systems fail to protect the activeagent (cargo); (6) the systems adversely alter the active agent; or (7)the systems fail to allow or promote absorption of the active agent.

[0007] Proteinoid microspheres have been used to deliverpharmaceuticals. See, for example, U.S. Pat. Nos. 5,401,516; 5,443,841;and Re. 35,862. In addition, certain modified amino acids have been usedto deliver pharmaceuticals. See, for example, U.S. Pat. Nos. 5,629,020;5,643,957; 5,766,633; 5,776,888; and 5,866,536.

[0008] More recently, a polymer has been conjugated to a modified aminoacid or a derivative thereof via a linkage group to provide forpolymeric delivery agents. The modified polymer may be any polymer, butpreferred polymers include, but are not limited to, polyethylene glycol(PEG), and derivatives thereof. See, for example, International PatentPublication No. WO 00/40203.

[0009] However, there is still a need for simple, inexpensive deliverysystems which are easily prepared and which can deliver a broad range ofactive agents by various routes.

SUMMARY OF THE INVENTION

[0010] The present invention provides compounds and compositions whichfacilitate the delivery of active agents. Delivery agent compounds ofthe present invention include those having the following formulas:

[0011] and salts thereof. Mixtures of these delivery agent compounds mayalso be used to facilitate the delivery of active agents.

[0012] The invention also provides a composition comprising at least oneof the delivery agent compounds of the formulas above, and at least oneactive agent. These compositions deliver active agents to selectedbiological systems in increased or improved bioavailability of theactive agent compared to administration of the active agent without thedelivery agent compound.

[0013] Also provided are dosage unit forms comprising the compositions.The dosage unit may be in the form of a liquid or a solid, such as atablet, capsule or particle, including a powder or sachet.

[0014] Another embodiment is a method for administering an active agentto an animal in need of the active agent, by administering a compositioncomprising at least one of the delivery agent compounds of the formulaabove and the active agent to the animal. Preferred routes ofadministration include the oral, intracolonic and pulmonary routes.

[0015] Yet another embodiment is a method of treating a disease or forachieving a desired physiological effect in an animal by administeringthe composition of the present invention.

[0016] Yet another embodiment is a method of preparing a composition ofthe present invention by mixing at least one delivery agent compound ofthe formula above, and at least one active agent.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Delivery Agent Compounds

[0018] The terms “alkyl” and “alkenyl” as used herein include linear andbranched alkyl and alkenyl substituents, respectively.

[0019] The delivery agent compounds may be in the form of the free baseor salts thereof. Suitable salts include, but are not limited to,organic and inorganic salts, for example, hydrochloride, ammonium,acetate, citrate, halide, hydroxide, sulfate, nitrate, phosphate,alkoxy, perchlorate, tetrafluoroborate, carboxylate, mesylate, fumerate,malonate, succinate, tartrate, acetate, gluconate, and maleate salts.The salts may also be solvates, including ethanol solvates, andhydrates. The mesylate salt can be formed by reacting the free base ofthe delivery agent compound with methanesulfonic acid.

[0020] Salts of the delivery agent compounds of the present inventionmay be prepared by methods known in the art. For example, citrate saltsmay be prepared in ethanol, toluene and citric acid. The salts may alsobe solvates, including ethanol solvates, and hydrates.

[0021] The delivery agent compound may be purified by recrystallizationor by fractionation on one or more solid chromatographic supports, aloneor linked in tandem. Suitable recrystallization solvent systems include,but are not limited to, ethanol, water, heptane, ethyl acetate,acetonitrile, methanol, and tetrahydrofuran (THF) and mixtures thereof.Fractionation may be performed on a suitable chromatographic supportsuch as alumina, using methanol/n-propanol mixtures as the mobile phase;reverse phase chromatography using trifluoroacetic acid/acetonitrilemixtures as the mobile phase; and ion exchange chromatography usingwater or an appropriate buffer as the mobile phase. When anion exchangechromatography is performed, preferably a 0-500 mM sodium chloridegradient is employed.

[0022] The delivery agent may contain a polymer conjugated to it by alinkage group selected from the group consisting of —NHC(O)NH—,—C(O)NH—, —NHC(O), —OOC—, —COO—, —NHC(O)O—, —OC(O)NH—, —CH₂NH —NHCH₂—,—CH₂NHC(O)O—, —OC(O)NHCH₂—, —CH₂NHCOCH₂O—, —OCH₂C(O)NHCH₂—,—NHC(O)CH₂O—, —OCH₂C(O)NH—, —NH—, —O—, and carbon-carbon bond, with theproviso that the polymeric delivery agent is not a polypeptide orpolyamino acid. The polymer may be any polymer including, but notlimited to, alternating copolymers, block copolymers and randomcopolymers, which are safe for use in mammals. Preferred polymersinclude, but are not limited to, polyethylene; polyacrylates;polymethacrylates; poly(oxyethylene); poly(propylene); polypropyleneglycol; polyethylene glycol (PEG); and derivatives thereof andcombinations thereof. The molecular weight of the polymer typicallyranges from about 100 to about 200,000 daltons. The molecular weight ofthe polymer preferably ranges from about 200 to about 10,000 daltons. Inone embodiment, the molecular weight of the polymer ranges from about200 to about 600 daltons and more preferably ranges from about 300 toabout 550 daltons.

[0023] Active Agents

[0024] Active agents suitable for use in the present invention includebiologically active agents and chemically active agents, including, butnot limited to, pesticides, pharmacological agents, and therapeuticagents. Suitable active agents include those that are rendered lesseffective, ineffective or are destroyed in the gastro-intestinal tractby acid hydrolysis, enzymes and the like. Also included as suitableactive agents are those macromolecular agents whose physiochemicalcharacteristics, such as, size, structure or charge, prohibit or impedeabsorption when dosed orally.

[0025] For example, biologically or chemically active agents suitablefor use in the present invention include, but are not limited to,proteins; polypeptides; peptides; hormones; polysaccharides, andparticularly mixtures of mucopolysaccharides; carbohydrates; lipids;small polar organic molecules (i.e. polar organic molecules having amolecular weight of 500 daltons or less); other organic compounds; andparticularly compounds which by themselves do not pass (or which passonly a fraction of the administered dose) through the gastro-intestinalmucosa and/or are susceptible to chemical cleavage by acids and enzymesin the gastrointestinal tract; or any combination thereof.

[0026] Further examples include, but are not limited to, the following,including synthetic, natural or recombinant sources thereof: growthhormones, including human growth hormones (hGH), recombinant humangrowth hormones (rhGH), bovine growth hormones, and porcine growthhormones; growth hormone releasing hormones; growth hormone releasingfactor, interferons, including α, β and γ; interleukin-1; interleukin-2;insulin, including porcine, bovine, human, and human recombinant,optionally having counter ions including zinc, sodium, calcium andammonium; insulin-like growth factor, including IGF-1; heparin,including unfractionated heparin, heparinoids, dermatans, chondroitins,low molecular weight heparin, very low molecular weight heparin andultra low molecular weight heparin; calcitonin, including salmon, eel,porcine and human; erythropoietin; atrial naturetic factor; antigens;monoclonal antibodies; somatostatin; protease inhibitors;adrenocorticotropin, gonadotropin releasing hormone; oxytocin;leutinizing-hormone-releasing-hormone; follicle stimulating hormone;glucocerebrosidase; thrombopoietin; filgrastim; prostaglandins;cyclosporin; vasopressin; cromolyn sodium (sodium or disodiumchromoglycate); vancomycin; desferrioxamine (DFO); bisphosphonates,including alendronate, tiludronate, etidronate, clodronate, pamidronate,olpadronate, and incadronate; parathyroid hormone (PTH), including itsfragments; anti-migraine agents such as BIBN-4096BS and other calcitoningene-related proteins antagonists; antimicrobials, includingantibiotics, anti-bacterials and anti-fungal agents; vitamins; analogs,fragments, mimetics or polyethylene glycol (PEG)-modified derivatives ofthese compounds; or any combination thereof. Non-limiting examples ofantibiotics include gram-positive acting, bacteriocidal, lipopeptidaland cyclic peptidal antibiotics, such as daptomycin and analogs thereof.

[0027] Delivery Systems

[0028] The composition of the present invention comprises one or moredelivery agent compounds of the present invention, and one or moreactive agents. In one embodiment, one or more of the delivery agentcompounds, or salts of these compounds, or poly amino acids or peptidesof which these compounds or salts form one or more of the units thereof,may be used as a delivery agent by mixing with the active agent prior toadministration to form an administration composition.

[0029] The administration compositions may be in the form of a liquid.The solution medium may be water (for example, for salmon calcitonin,parathyroid hormone, and erythropoietin), 25% aqueous propylene glycol(for example, for heparin) and phosphate buffer (for example, for rhGH).Other dosing vehicles include polyethylene glycol. Dosing solutions maybe prepared by mixing a solution of the delivery agent compound with asolution of the active agent, just prior to administration. Alternately,a solution of the delivery agent compound (or active agent) may be mixedwith the solid form of the active agent (or delivery agent compound).The delivery agent compound and the active agent may also be mixed asdry powders. The delivery agent compound and the active agent can alsobe admixed during the manufacturing process.

[0030] The dosing solutions may optionally contain additives such asphosphate buffer salts, citric acid, glycols, or other dispersingagents. Stabilizing additives may be incorporated into the solution,preferably at a concentration ranging between about 0.1 and 20% (w/v).

[0031] In one embodiment, dosing solution containing BIBN-4096BS have apH of less than 8. According to another embodiment, dosing solutionscontaining BIBN-4096BS have a pH of less than 7.

[0032] The administration compositions may alternately be in the form ofa solid, such as a tablet, capsule or particle, such as a powder orsachet. Solid dosage forms may be prepared by mixing the solid form ofthe compound with the solid form of the active agent. Alternately, asolid may be obtained from a solution of compound and active agent bymethods known in the art, such as freeze-drying (lyophilization),precipitation, crystallization and solid dispersion.

[0033] The administration compositions of the present invention may alsoinclude one or more enzyme inhibitors. Such enzyme inhibitors include,but are not limited to, compounds such as actinonin or epiactinonin andderivatives thereof. Other enzyme inhibitors include, but are notlimited to, aprotinin (Trasylol) and Bowman-Birk inhibitor.

[0034] The amount of active agent used in an administration compositionof the present invention is an amount effective to accomplish thepurpose of the particular active agent for the target indication. Theamount of active agent in the compositions typically is apharmacologically, biologically, therapeutically, or chemicallyeffective amount. However, the amount can be less than that amount whenthe composition is used in a dosage unit form because the dosage unitform may contain a plurality of delivery agent compound/active agentcompositions or may contain a divided pharmacologically, biologically,therapeutically, or chemically effective amount. The total effectiveamount can then be administered in cumulative units containing, intotal, an effective amount of the active agent.

[0035] The total amount of active agent to be used can be determined bymethods known to those skilled in the art. However, because thecompositions of the invention may deliver active agents more efficientlythan compositions containing the active agent alone, lower amounts ofbiologically or chemically active agents than those used in prior dosageunit forms or delivery systems can be administered to the subject, whilestill achieving the same blood levels and/or therapeutic effects.

[0036] The presently disclosed delivery agent compounds facilitate thedelivery of biologically and chemically active agents, particularly inoral, intranasal, sublingual, intraduodenal, subcutaneous, buccal,intracolonic, rectal, vaginal, mucosal, pulmonary, transdermal,intradermal, parenteral, intravenous, intramuscular and ocular systems,as well as traversing the blood-brain barrier.

[0037] Dosage unit forms can also include any one or combination ofexcipients, diluents, disintegrants, lubricants, plasticizers,colorants, flavorants, taste-masking agents, sugars, sweeteners, salts,and dosing vehicles, including, but not limited to, water, 1,2-propanediol, ethanol, olive oil, or any combination thereof.

[0038] The compounds and compositions of the subject invention areuseful for administering biologically or chemically active agents to anyanimals, including but not limited to birds such as chickens; mammals,such as rodents, cows, pigs, dogs, cats, primates, and particularlyhumans; and insects.

[0039] The system is particularly advantageous for delivering chemicallyor biologically active agents that would otherwise be destroyed orrendered less effective by conditions encountered before the activeagent reaches its target zone (i.e. the area in which the active agentof the delivery composition is to be released) and within the body ofthe animal to which they are administered. Particularly, the compoundsand compositions of the present invention are useful in orallyadministering active agents, especially those that are not ordinarilyorally deliverable, or those for which improved delivery is desired.

[0040] The compositions comprising the compounds and active agents haveutility in the delivery of active agents to selected biological systemsand in an increased or improved bioavailability of the active agentcompared to administration of the active agent without the deliveryagent. Delivery can be improved by delivering more active agent over aperiod of time, or in delivering active agent in a particular timeperiod (such as to effect quicker or delayed delivery), or in deliveringthe active agent at a specific time, or over a period of time (such assustained delivery).

[0041] Another embodiment of the present invention is a method for thetreatment or prevention of a disease or for achieving a desiredphysiological effect, such as those listed in the table below, in ananimal by administering the composition of the present invention.Specific indications for active agents can be found in the Physicians'Desk Reference (54^(th) Ed., 2000, Medical Economics Company, Inc.,Montvale, N.J.), which is herein incorporated by reference. The activeagents in the table below include their analogs, fragments, mimetics,and polyethylene glycol-modified derivatives. Active Agent Disease andPhysiological Effect Growth hormones Growth disorders Interferons,including α, β and γ. Viral infection, including chronic cancer andmultiple sclerosis Interleukin-1; interleukin-2. Viral infection; cancerInsulin; Insulin-like growth factor Diabetes IGF-1. Heparin Thrombosis;prevention of blood coagulation Calcitonin. Osteoporosis; diseases ofthe bone Erythropoietin Anemia Atrial naturetic factor VasodilationAntigens Infection Monoclonal antibodies To prevent graft rejection;cancer Somatostatin Bleeding ulcer; erosive gastritis Proteaseinhibitors AIDS Adrenocorticotropin High cholesterol (to lowercholesterol) Gonadotropin releasing hormone Ovulatory disfunction (tostimulate ovulation) Oxytocin Labor disfunction (to stimulatecontractions) Leutinizing-hormone-releasing- Regulate reproductivefunction hormone; follicle stimulating hormone GlucocerebrosidaseGaucher disease (to metabolize lipoprotein) ThrombopoietinThrombocytopenia Filgrastim Reduce infection in chemotherapy patientsProstaglandins Hypertension Cyclosporin Transplant rejection VasopressinBed-wetting; antidiuretic Cromolyn sodium; Vancomycin Asthma; allergiesDesferrioxamine (DFO) Iron overload Parathyroid hormone (PTH),Osteoporosis; including its fragments. Diseases of the boneAntimicrobials Infection including gram- positive bacterial infectionVitamins Vitamin deficiencies Bisphosphonates Osteoporosis; Paget'sdisease; Inhibits osteoclasts BIBN4096BS - (1- Anti-migraine; calcitoningene- Piperidinecarboxamide. N-[2-[[5- related peptide antagonistamino-1-[[4-(4-pyridinyl)-1- piperazinyl)carbonyl]pentyl]amino]-1-[(3,5-dibromo-4- hydroxyphenyl)methyl]-2-oxoethyl]-4(1,4-dihydro-2-oxo-3(2H0- quinazolinyl)-.[R-(R*,S*)]-)

[0042] For example, one embodiment of the present invention is a methodfor treating a patient suffering from or susceptible to diabetes byadministering insulin and at least one of the delivery agent compoundsof the present invention.

[0043] Following administration, the active agent present in thecomposition or dosage unit form is taken up into the circulation. Thebioavailability of the agent is readily assessed by measuring a knownpharmacological activity in blood, e.g. an increase in blood clottingtime caused by heparin, or a decrease in circulating calcium levelscaused by calcitonin. Alternately, the circulating levels of the activeagent itself can be measured directly.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The following examples illustrate the invention withoutlimitation. All parts are given by weight unless otherwise indicated.

[0045] Proton nuclear magnetic resonance (¹H NMR) analyses for thecompounds listed below were conducted on a 300 MHz Bruker spectrometerusing dimethyl sulfoxide (DMSO-d₆) as the solvent unless otherwiseindicated.

[0046] Liquid chromatograph/mass spectrometry (LC-MS) analyses wereperformed with an Agilent Technologies, LC/MSD 1100 (single quad) havingthe following parameters:

[0047] Mobile Phase A: 50:950:5 acetonitrile:water:acetic acid (v/v/v)

[0048] Mobile Phase B: 950:50:5 acetonitrile:water:acetic acid (v/v/v)

[0049] Gradient Elution: 4 minute linear gradient 0-100% B; total timeper injection is 11 minutes

[0050] Injection volume: 5 uL

[0051] Column: ZORBAX Rapid Resolution Cartridge, SB-C18, 2.1×30 mm, 3.5um

[0052] Particle size, catalog # 873700-902

[0053] Column temp: 40° C.

[0054] UV detection at 244 nm

[0055] MSD parameters:

[0056] Source: API-ES, positive polarity

[0057] Scan Parameters:

[0058] Mass Range: 125.00-600.00

[0059] Fragmentor: 60 V

[0060] Gain: 1.0 EMV

[0061] Threshold: 150

[0062] Spray Chamber:

[0063] Gas Temp. 350 deg. D

[0064] Drying Gas: 12.0 l/min

[0065] Neb. Pressure; 40 psig

[0066] VCap 4000V positive/negative

EXAMPLE 1 Preparation of Compounds

[0067] 1a: Preparation of Compound 2

[0068] A solution of 40% dimethylamine/water (30 mL, 26.9 g, 239 mmol)and ethanol (50 mL) was treated with a solution of 8-bromo-1-octanol(15.13 g, 72.3 mmol) and ethanol (20 mL), added dropwise over 10minutes. The reaction mixture was stirred for 75 hours, diluted withethyl acetate (80 mL) and washed with saturated sodium bicarbonatesolution. The aqueous phase was extracted with ethyl acetate. Thecombined organic phases were washed with saturated sodium bicarbonatesolution (50 mL) and brine (2×40 mL), dried over sodium sulfate, andconcentrated. The 11.4 g of 8-dimethylamino-1-octanol, isolated as ayellow oil, was used as is.

[0069] A slurry of carsalam (10.80 g, 66.2 mmol),8-dimethylamino-1-octanol (11.4 g, 65.8 mmol), triphenylphosphine (17.53g, 66.8 mmol) and tetrahydrofuran (40 mL) was treated with a solution ofdiisopropyl azodicarboxylate (13.0 mL, 13.35 g, 66.0 mmol) andtetrahydrofuran (20 mL,), added dropwise over 25 minutes causing thetemperature to rise to 50° C. The reaction mixture was allowed to coolback to 250 C and stirred for 40 hours. The solution was treated withaqueous 2N NaOH (70 mL, 140 mmol) and warmed to 600 C for 180 minutes.The cooled reaction mixture was washed with ethyl acetate (2×50 mL). Theaqueous phase was acidified with 4% aqueous HCl to a pH slightly lessthan 0 and was washed with ethyl acetate (2×40 mL). The pH of theaqueous phase was raised to 9.5 upon treatment with solid sodiumbicarbonate. The aqueous phase was extracted with methylene chloride(10×40 mL). The combined methylene chloride extracts were dried oversodium sulfate and concentrated to give 0.6 g of product. The rest ofthe product was found in the earlier ethyl acetate extracts. These ethylacetate layers were extracted with 1N aqueous NaOH (4×30 mL). These 4aqueous phases were combined, acidified to pH 0.7 with 4% aqueous HCl,and washed with ethyl acetate (2×30 mL). The pH of the aqueous solutionwas adjusted to 5 with aqueous 2N NaOH. The solution was treated withsolid sodium bicarbonate until no more bubbling occurred and wasextracted with ethyl acetate (6×50 mL). The combined ethyl acetatelayers were dried over sodium sulfate and concentrated to a solid. Thesolid was taken up into a tetrahydrofuran and treated with HCl gas.Water was added causing a solid to precipitate out of solution. A totalof 4.73 g of N-(8-dimethylaminooctyl)salicylamide hydrochloride wasisolated by filtration.

[0070] m.p. 78-80° C.; ¹H NMR (DMSO-d₆), δ (ppm): 10.5 (bs, 1H), 8.9 (t,1H), 7.9 (dd, 1H), 7.4 (td, 1H), 6.9 (m, 2H), 3.3 (q, 2H), 3.0 (m, 2H),2.7 (s, 6H), 1.6 (t, 4H), 1.3 (m, 8H). KF value=5.19% water. Elementalanalysis: % C, 58.86 (calculated), 58.87 (found); % H, 9.01(calculated), 8.98 (found); % N, 8.08 (calculated), 7.98 (found).

[0071] 1b: Preparation of Compound 4

[0072] A one-neck, 250 mL round bottomed flask was charged with4-(dimethylamino)benzylamine dihydrochloride (8.0 g, 0.0358 mol) and 50mL of methylene chloride. The stirred solution was chilled in an icebath. Triethylamine (20.0 mL, 0.1432 mol) and a catalytic amount of4-(dimethylamino)pyridine (DMAP) were added to the reaction mixture. Asolution of acetylsalicyloyl chloride (7.12 g, 0.1432 mol) in methylenechloride (30 mL) was added to the chilled reaction flask. After theaddition was complete, the reaction mixture was warmed to roomtemperature and was allowed to stir overnight.

[0073] The reaction mixture was diluted with 2N HCl, and the layers wereseparated. The organic phase was washed with water, dried over sodiumsulfate, and concentrated in vacuo.

[0074] The resulting oil was stirred in 2N NaOH for 4 hours, and washedwith ethyl acetate. The aqueous phase was concentrated in vacuo toremove any residual ethyl acetate. The pH of the aqueous phase wasadjusted to 7, and the resulting solids were collected by vacuumfiltration. The weight of the crude solids was 1.76 g. The reaction wasrepeated to give an additional 0.88 g of crude product. The combinedsolids were recrystallized from ethanol/water to give 2.64 g ofN-(4-dimethylaminobenzyl) salicylamide as a white solid.

[0075] m.p. 129-132° C.; ¹H NMR (d₆-DMSO), δ (ppm): 9.3 (s, 1H), 7.87(dd, 1H), 7.39 (dt, 1H), 7.16 (d, 2H), 6.87 (t, 2H), 6.69 (d, 2H), 4.38(d, 2H), 2.86 (s, 6H). Elemental analysis: % C, 71.09 (calc.), 70.84(found); % H, 6.71 (calc.), 6.50 (found); % N, 10.36 (calc.), 10.14(found).

[0076] 1c: Preparation of Compound 5

[0077] A suspension of morpholine (3.30 mL, 3.30 g, 37.8 mmol),6-bromo-1-hexanol (6.72 g, 72.3 mmol), ethanol (20 mL), and potassiumcarbonate (6.23 g, 45.1 mmol) was stirred for 36 hours at 25° C., aftera slight exotherm at the beginning. The reaction mixture was dilutedwith ethyl acetate (30 mL), filtered and concentrated. The residue wastaken up in ethyl acetate, filtered and concentrated. The 7.0 g of4-(6-hydroxyhexyl)morpholine was isolated as a yellow oil and used asis.

[0078] A slurry of carsalam (6.13 g, 37.6 mmol),4-(6-hydroxyhexyl)morpholine (7.0 g, 37.4 mmol), triphenylphosphine(9.97 g, 35.0 mmol), and tetrahydrofuran (40 mL) was treated with asolution of diisopropyl azodicarboxylate (7.40 mL, 7.60 g, 37.6 mmol)and tetrahydrofuran (10 mL), added dropwise over 15 minutes. Thereaction mixture was stirred at 25° C. for 60 hours. The solution wastreated with aqueous 2N NaOH (50 mL, 100 mmol) and warmed to 60° C. for180 minutes. The cooled reaction mixture was concentrated to remove thetetrahydrofuran. The aqueous residue was washed with ethyl acetate (2×40mL), and was acidified with 4% aqueous HCl to a pH of 0.84 (causingcarbon dioxide gas to evolve). The pH of the aqueous phase was raised to7.8 with 2N aqueous NaOH. Solid sodium bicarbonate was added. Extractionwith ethyl acetate (8×40 mL), drying over sodium sulfate andconcentration gave an oil which solidified upon standing. A total of4.26 g of 4-(6-morpholin-4-ylhexyl)-salicylamide was isolated.

[0079] m.p. 70-73° C.; ¹H NMR (d₆-DMSO), δ (ppm): 8.8 (t, 1H), 7.8 (dd,1H), 7.4 (td, 1H), 6.9 (m, 2H), 3.5 (t, 4H), 3.3 (q, 2H), 2.3 (t, 4H),2.2, (t, 2H), 1.5 (t, 2H), 1.3-1.4 (m, 6H). KF value=5.39% water.Elemental analysis: % C, 63.05 (calculated), 63.06 (found); % H, 8.70(calculated), 8.53 (found); % N, 8.65 (calculated), 8.73 (found).

[0080] 1d: Preparation of the Citrate Salts of Compounds 1,7

[0081] Synthesis of N-Hydroxysuccimide-O-acetyl-5-chlorosalicylate

[0082] 5-Chlorosalicylic acid (17.3 g, 100 mmol) and three drops ofconcentrated sulfuric acid (98%) were added to a solution of aceticanhydride (14 g, 137 mmol) and glacial acetic acid (16.4 g, 274 mmol)with stirring. The reaction mixture was slowly heated to 70° C. andstirred for 2 hours. The reaction mixture was cooled to room temperatureand was gradually added to ice water (500 mL) to precipitate theacetylated product. These solids were collected and washed with water.The two batches of product were combined and recrystallized in ethylacetate. The pure crystals were collected via vacuum filtration to give16.3 g of O-acetyl-5-chlorosalicylic acid (76 mmol, 76% yield).

[0083] Elemental analysis calculated for C₉H₇O₄Cl: C, 50.37%, H, 3.29%;N, 0.0%; Found: C, 50.36%; H, 3.20%; N, <0.02%.

[0084] N-Hydroxysuccinimide (8.6 g, 82 mmol) was dissolved in dimethylformamide (DMF) (8 mL). This solution was mixed withO-Acetyl-5-chlorosalicylic acid (16 g, 74.6 mmol) in dichloromethane(DCM) (150 mL) at room temp. This mixture was stirred in a water bath.1,3-dicyclohexylcarbodiimide (DCC) (17 g, 82 mmol) was dissolved indichloromethane (55 mL) and was gradually added to the mixture. Thereaction equilibrated to room temperature and stirred for 24 hours. Themixture was cooled to −10° C. and was filtered to remove any solids. Thefiltrate was diluted with dichloromethane (100 mL). The solution waswashed with 1N HCl (2×200 mL), brine (2×200 mL), 5% sodium bicarbonate(2×200 mL) and brine (2×200 mL). The dichloromethane layer was driedover anhydrous sodium sulfate. The solvent was evaporated in vacuo. Theproduct was collected to give 22.5 g (72 mmol, 97%).

[0085] Compound 7:2-(5-chloro-2-hydroxybenzoyl)amino-5-(N′,N′-diethylamino)pentaneMono-Citrate

[0086] IUPAC name:N-[5-(diethylamino)-1-methylpentyl]-5-chloro-2-hydroxybenzamideMono-Citrate

[0087] N-Hydroxysuccimide-O-acetyl-5-chlorosalicylate obtained above(3.1 g, 10 mmol) was dissolved in dichloromethane (15 mL). This solutionwas slowly added to 2-amino-5-diethylaminopentane (3.2 g, 20 mmol) indichloromethane (35 mL) with stirring. The reaction mixture was stirredovernight at room temperature. HPLC indicated the completion of thereaction. The reaction mixture was washed with 5% sodium bicarbonate(3×100 mL). The organic layer was dried over anhydrous sodium sulfate.The dichloromethane evaporated in vacuo to give the free tertiary amineproduct (2.3 g, 7.4 mmol, 74%).

[0088] The above obtained2-(5-chloro-2-hydroxybenzoylamino)-5-(N,N-diethylamino)pentane (2.3 g,7.4 mmol) and citric acid (1.4 g, 7.4 mmol) were dissolved in anhydrousethyl alcohol (8 mL). Diethylether (˜30 mL) was added to this solutionuntil the solution became cloudy. The cloudy solution was refrigeratedovernight to precipitate the citric acid salt. The final product,2-(5-chloro-2-hydroxybenzoylamino)-5-(N,N-diethylamino)pentanemono-citrate, was collected via vacuum filtration and dried undernitrogen flow to give 2.0 g (4.0 mmol, 54%).

[0089] m.p. 57-59° C.; ¹HNMR (DMSO-d₆) δ (ppm): 1.18 (m, 9H), 1.60 (m,4H), 2.57 (q_(ab), 4H), 3.08 (m, 6H), 4.04 (q, 1H), 4.11 (s, 1H), 6.98(d, 1H), 7.47 (dd, 1H), 8.01 (d, 1H), 8.70 (s, 1H). KF value=0.59%.Elemental analysis for calculated C₂₂H₃₃N₂O₉Cl: C, 52.33; H, 6.54; N,5.55. Found: C, 52.21; H, 6.79; N, 5.20.

[0090] Compound 1: N-(5-chloro-2-hydroxybenzoyl)-N′,N′-diethylenediamineMono-Citrate

[0091] IUPAC name: N-[2-(diethylamino)ethyl]-5-chloro-2-hydroxybenzamideMono-Citrate

[0092] Compound 1 was prepared by the same procedure as for compound 7with the appropriate starting materials.

[0093] m.p. 107-109° C.; ¹HNMR (DMSO-d₆), δ (ppm): 1.16 (t, 6H), 2.61(q_(ab), 4H), 3.09 (m, 6H), 3.59 (s, 2H), 6.98 (d, 1H), 7.44 (dd, 1H),7.88 (d, 1H), 9.09 (s, 1H), 9.95-11.20 (s, 3H).

[0094] Elemental analysis for calculated C₁₉H₂₇N₂O₉Cl: C 49.30, H, 5.84;N, 6.05; found: C, 49.30; H, 5.78; N, 5.94.

[0095] 1e: Preparation of Compound 11,18

[0096] Synthesis of N-Hydroxysuccimide-O-acetyl-4-methoxysalicylate

[0097] N-hydroxysuccimide-O-acetyl-4-methoxysalicylate was prepared bythe same procedure as for N-hydroxy-O-acetyl-5-chlorosalicylate with theappropriate starting materials.

[0098] Compound 11: 8-(2-hydroxy-4-methoxybenzoylamido) OctylamineHydrogen Chloride

[0099] IUPAC name: N-(8-aminooctyl)-2-hydroxy-4-methoxybenzamideHydrogen Chloride

[0100] N-hydroxysuccimide-O-acetyl-4-methoxysalicylate as obtained above(13 g, 42.3 mmol) was dissolved in dichloromethane (70 mL) and addeddropwise to a solution of 1,8-diaminooctane (13 g, 90 mmol) indichloromethane (230 mL) in a water bath with stirring. The reaction wasstirred overnight. The product precipitated out of solution and wascollected via vacuum filtration. The precipitates were washed withdichloromethane and were air-dried to give 9.0 g of crude product. Theprecipitates were washed with water (50 mL) and extracted with 0.1N HClaqueous solution (50 mL) for 0.5 hours with stirring. The acidic aqueoussolution was filtered to remove insoluble material. The filtrate waswashed with ethyl ether (150 mL) and was adjusted to pH 10.Precipitation occurred immediately. The mixture was allowed to stand atroom temperature over night. The precipitate was collected by filtrationand air-dried to yield 2.6 g (8.8 mmol, 21%).

[0101] The free primary amine obtained above (1.7 g, 5.8 mmol) wassuspended in 20 mL of anhydrous ethyl alcohol. HCl gas was bubbled intothis mixture for 10 minutes to obtain a clear solution. Nitrogen gas wasbubbled through this solution to purge the excess HCl and to evaporatethe ethyl alcohol until the volume of the solution was 10 mL. Thissolution was refrigerated for 2 hours to precipitate the product. Theproduct was collected via vacuum filtration, was washed with ethyl etherand dried in vacuo to give 1.7 g of the hydrochloric salt (5.1 mmol,89%).

[0102] m.p. 162-164° C.; ¹HNMR (DMSO-d₆), 6(ppm): 1.36 (s, 8H), 1.53 (m,4H), 2.71 (sex, 2H), 3.22 (q, 2H), 3.76 (s, 3H), 6.42 (m, 2H), 7.83 (d,1H), 7.93-8.11 (s, 3H), 8.77 (t, 1H), 13.12 (bs, 1H). Elemental analysisfor calculated C₁₆H₂₇N₂O₃Cl: C, 58.08; H, 8.23; N, 8.47; found: C,57.46; H, 8.24; N, 8.63.

[0103] Compound 18: N-(2-hydroxy-4-methoxybenzoyl)-1,8-diaminooctane

[0104] IUPAC name:N-[2-(diethylamino)ethyl]-2-hydroxy-4-methoxybenzamide

[0105] Compound 18 was prepared by the same procedure as for compound 11with the appropriate starting materials.

[0106] m.p. 151-153° C.; ¹HNMR (DMSO-d₆), δ (ppm): 1.16-1.40 (m, 8H),1.46 (m, 4H), 2.64 (t, 2H), 3.23 (s, 2H), 3.69 (s, 3H), 6.13 (d, 1H),6.17 (s, 1H), 7.67 (d, 1H), 10.05 (s, 1H). KF value=0.93%. Elementalanalysis for calculated C₁₆H₂₆N₂O₃O₁₆H₂O: C, 64.67; H, 8.86; N, 9.43;found: C, 64.26; H, 8.84; N, 9.65.

[0107] 1f: Preparation of Citrate Salts of Compounds 3,6,8 and Compounds9,10,17

[0108] Synthesis of N-Hydroxysuccimide-O-acetylsalicylate

[0109] N-Hydroxysuccinimide (12 g, 104 mmol) was dissolved in DMF (15mL). This solution was mixed with O-acetylsalicoyl chloride (20 g, 101mmol) in dichloromethane (150 mL) at room temperature. Triethylamine (11g, 109 mmol) was added dropwise to this mixture with stirring. Thereaction mixture was stirred for 2 hours. The mixture was filtered toremove any insoluble material. The filtrate was collected and thesolvents were evaporated in vacuo. The resulting oil was dissolved in200 mL of ethyl acetate. Any remaining solids were removed byfiltration. The filtrate was washed with 1N HCl (3×150 mL), brine (1×150mL), 4% sodium bicarbonate (3×150 mL) and brine (1×150 mL). The ethylacetate layer was dried over anhydrous sodium sulfate. Ethyl acetate wasremoved by vacuum evaporation, followed by a nitrogen purge. 20 g (72mmol, 72%) of N-hydroxysuccimide-O-acetylsalicylate was produced.

[0110] Compound 3: N-(2-hydroxybenzoyl)-N′,N′-diethylenediamineMono-Citrate

[0111] IUPAC name: N-[2-(diethylamino)ethyl]-2-hydroxybenzamideMono-Citrate

[0112] Compound 3 was prepared by the same procedure as for compound 7with the appropriate starting materials.

[0113] m.p. 111-113° C.; ¹HNMR (DMSO-d₆), δ (ppm): 1.17 (t, 6H), 2.59(q_(ab), 4H), 3.09 (m, 6H), 3.61 (q, 2H), 6.95 (m, 2H), 7.44 (t, 1H),7.85 (d, 1H), 9.07 (s, 1H), 10.25 (bs, 2H). KF value=0.30%. Elementalanalysis for calculated Cl₉H₂₈N₂O₉: C, 53.27; H, 6.54; N, 6.54; found:C, 52.96; H, 6.28; N, 6.37.

[0114] 2-(2-hydroxybenzoyl)amino-5-(N,N-diethylamino)pentaneMono-Citrate

[0115] IUPAC name:N-[5-(diethylamino)-1-methylpentyl]-2-hydroxybenzamide Mono-Citrate

[0116] 2-(2-hydroxybenzoyl)amino-5-(N,N-diethylamino)pentanemono-citrate was prepared by the same procedure as for compound 7 withthe appropriate starting materials.

[0117] m.p. 62-64° C.; ¹HNMR (DMSO-d₆), δ (ppm): 1.18 (m, 9H), 1.61 (m,4H), 2.57 (q_(ab), 4H), 3.02 (m, 6H), 4.03 (q, 1H), 4.11 (s, 1H), 6.91(m, 2H), 7.41 (t, 1H), 7.89 (d, 1H), 8.58 (s, 1H), 10.611.8 (s, 2H).Elemental analysis for calculated C₂₂H₃₄N₂O₉: C, 56.17; H, 7.23; N,5.96; found: C, 55.77; H, 7.35; N, 5.71.

[0118] Compound 8:N-(2-hydroxybenzoyl)-N′,N′-di(n-butyl)-1,3-diaminonopropane Mono-Citrate

[0119] IUPAC name:N-{3-[dibutylamino]propyl}-5-chloro-2-hydroxybenzamide Mono-Citrate

[0120] The procedures were the same as those described for Compound 7except for the starting materials.

[0121] m.p. 87-89° C.; ¹HNMR (DMSO-d₆), δ (ppm): 0.89 (t, 6H), 1.30(sex, 4H), 1.53 (m, 4H), 1.77 (quin, 2H), 2.59 (q_(ab), 4H), 2.843.04(m, 6H), 3.36 (t, 2H), 6.96 (d, 1H), 7.44 (dd, 1H), 7.90 (d, 1H), 8.97(s, 1H). Elemental analysis for calculated C₁₉H₂₇N₂O₉Cl: C, 54.08; H,6.95; N, 5.26; found: C, 54.13; H, 7.00; N, 5.10.

[0122] Compound 9: N-(2-hydroxybenzoyl)-1,12-diaminododecane HydrogenChloride

[0123] IUPAC name: N-(12-aminododecanyl)-2-hydroxybenzamide HydrogenChloride

[0124] Compound 9 was prepared by the same procedure as for compound 7with the appropriate starting materials.

[0125] m.p. 140-142° C.; ¹HNMR (DMSO-d₆), δ (ppm): 1.21 (s, 16H), 1.53(m, 4H), 2.72 (sex, 2H), 3.27 (q, 2H), 6.89 (m, 2H), 7.37 (t, 1H),7.7.91 (d, 1H), 7.96-8.20 (s, 3H), 8.93 (s, 1H), 12.87 (s, 1H).Elemental analysis for calculated C₁₉H₃₃N₂O₂Cl: C, 63.94; H, 9.32; N,7.85; Cl, 9.93; found: C, 63.33; H, 9.45; N, 7.28; Cl, 10.87.

[0126] Compound 10: 10-(2-hydroxybenzoylamido) Decylamine HydrogenChloride

[0127] IUPAC name: N-(10-aminodecyl)-2-hydroxybenzamide HydrogenChloride

[0128] Compound 10 was prepared by the same procedure as for compound 11with the appropriate starting materials.

[0129] m.p. 136-138° C.; ¹HNMR (DMSO-d₆), δ (ppm): 1.24 (s, 12H), 1.51(m, 4H), 2.71 (t, 2H), 3.26 (q, 2H), 6.87 (m, 2H), 7.37 (t, 1H), 7.88(d, 1H), 7.89-8.13 (s, 3H), 8.91 (t, 1H), 12.76 (s, 1H). Elementalanalysis for calculated C₁₇H₂₉N₂O₂Cl: C, 62.09; H, 8.89; N, 8.52; found:C, 60.66; H, 9.11; N, 8.73.

[0130] Compound 17: N-(2-hydroxybenzoyl)-1,9-diaminononane

[0131] IUPAC name: N-(8-aminononyl)-2-hydroxybenzamide

[0132] Compound 17 was prepared by the same procedure as for preparingthe free amine of compound 11 with the appropriate starting materials.

[0133]¹HNMR (DMSO-d₆), δ (ppm): 1.21-1.42 (m, 12H), 1.51 (m, 2H), 2.60(t, 2H), 3.27 (t, 2H), 6.63 (t, 1H), 6.70 (d, 1H), 7.22 (t, 1H), 7.78(d, 1H), 9.80 (s, 1H). KF value=0.91%. Elemental analysis for calculatedC₁₆H₂₆N₂O₂ (0.91% H₂O): C, 68.61; H, 9.33; N, 9.97; Found: C, 68.54; H,9.41; N, 10.31.

[0134]1g: Preparation of Compound 12

[0135] A 20 mL scintillation vial was charged with 3,5-dibromosalicylicacid (1.299 g, 4.39 mmol),1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide*HCl (0.99 g, 5.2 mmol)and 1-hydroxybenzotriazole hydrate (0.79 g, 5.8 mmol).1-(3-Aminopropyl)imidazole (476 ul, 3.99 mmol) was added by autopipet.THF (10 mL) was added, the vial was capped, and placed on an orbitalshaker overnight at 60° C. The heat was turned off and the vial wasallowed to cool back to room temperature. Trisamine resin (200 mg, 0.85mmol) was added and the vial placed back on the orbital shaker for 4hours. Amberlyst-15 (2 g, 9.4 mmol) and Amberlyst-21 (2 g, 9.4 mmol)ion-exchange resins were added to the vial along with DCM (5 mL) tosuspend the resins. The vial was placed back on the orbital shakerovernight. The reaction mixture was filtered and the resins were rinsedwith DCM (2×5 mL). The combined filtrates were placed under a nitrogenstream overnight. 2.3985 g of material was recovered.

[0136] LC-MS: rt=2.39 min, 89%, M+H=404

[0137] 1h: Preparation of Compound 13

[0138] A 20 mL scintillation vial was charged with 3,5-dichlorosalicylicacid (0.9082 g, 4.39 mmol),1-[3(dimethylamino)propyl]-3-ethylcarbodiimide*HCl (0.99 g, 5.2 mmol)and 1-hydroxybenzotriazole hydrate (0.79 g, 5.8 mmol).1-(3-Aminopropyl)imidazole (476 μl, 3.99 mmol) was added by autopipet.THF (10 mL) was added, the vial was capped, and placed on an orbitalshaker overnight at 60° C. The heat was turned off and the vial wasallowed to cool back to room temperature. Trisamine resin (200 mg, 0.85mmol) was added and the vial placed back on the orbital shaker for 4hours. Amberlyst-15 (2 g, 9.4 mmol) and Amberlyst-21 (2 g, 9.4 mmol)ion-exchange resins were added to the vial along with DCM (5 mL) tosuspend the resins. The vial was placed back on the orbital shakerovernight. The reaction mixture was filtered and the resins were rinsedwith DCM (2×5 mL). The combined filtrates were placed under a nitrogenstream overnight. 2.0343 g of material was recovered.

[0139] LC-MS: rt 2.24 min, 79%, M+H=315

[0140] 1i: Preparation of Compound 14

[0141] A 20 mL scintillation vial was charged with 3,5-diiodosalicylicacid (1.700 g, 4.39 mmol),1-[3(dimethylamino)propyl]-3-ethylcarbodiimide*HCl (0.99 g, 5.2 mmol)and 1-hydroxybenzotriazole hydrate (0.79 g, 5.8 mmol).1-(3-Aminopropyl)imidazole (476 μl, 3.99 mmol) was added by autopipet.THF (10 mL) was added, the vial was capped, and placed on an orbitalshaker overnight at 60° C. The heat was turned off and the vial wasallowed to cool back to room temperature. Trisamine resin (200 mg, 0.85mmol) was added and the vial placed back on the orbital shaker for 4hours. Amberlyst-15 (2 g, 9.4 mmol) and Amberlyst-21 (2 g, 9.4 mmol)ion-exchange resins were added to the vial along with DCM (5 mL) tosuspend the resins. The vial was placed back on the orbital shakerovernight. The reaction mixture was filtered and the resins were rinsedwith DCM (2×5 mL). The combined filtrates were placed under a nitrogenstream overnight. 2.7305 g of material was recovered.

[0142] LC-MS: rt=2.62 min, 84%, M+H=498

[0143] 1j: Preparation of Compound 15

[0144] A 20 mL scintillation vial was charged with 3,5-dibromosalicylicacid (1.299 g, 3.8 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide*HCl (0.84 g, 4.39 mmol) and1-hydroxybenzotriazole hydrate (0.59 g, 4.38 mmol).4-(3-Aminopropyl)morpholine (506 p1, 3.47 mmol) was added by autopipet.THF (10 mL) was added, the vial was capped, and placed on an orbitalshaker overnight at 60° C. The heat was turned off and the vial wasallowed to cool back to room temperature. Trisamine resin (200 mg, 0.85mmol) was added and the vial placed back on the orbital shaker for 4hours. Amberlyst-15 (2 g, 9.4 mmol) and Amberlyst-21 (2 g, 9.4 mmol)ion-exchange resins were added to the vial along with DCM (5 mL) tosuspend the resins. The vial was placed back on the orbital shakerovernight. The reaction mixture was filtered and the resins were rinsedwith DCM (2×5 mL). The combined filtrates were placed under a nitrogenstream overnight. 2.103 g of material was recovered.

[0145] LC-MS: rt=2.36 min, 74%, M+H=423

[0146] 1k: Preparation of Compound 16

[0147] A 20 mL scintillation vial was charged with 3,5-dichlorosalicylicacid (0.786 g, 3.8 mmol),1-[3(dimethylamino)propyl]-3-ethylcarbodiimide*HCl (0.84 g, 4.39 mmol)and 1-hydroxybenzotriazole hydrate (0.59 g, 4.38 mmol).4-(3-Aminopropyl)morpholine (506 μl, 3.47 mmol) was added by autopipet.THF (10 mL) was added, the vial was capped, and placed on an orbitalshaker overnight at 60° C. The heat was turned off and the vial wasallowed to cool back to room temperature. Trisamine resin (200 mg, 0.85mmol) was added and the vial placed back on the orbital shaker for 4hours. Amberlyst-15 (2 g, 9.4 mmol) and Amberlyst-21 (2 g, 9.4 mmol)ion-exchange resins were added to the vial along with DCM (5 mL) tosuspend the resins. The vial was placed back on the orbital shakerovernight. The reaction mixture was filtered and the resins were rinsedwith DCM (2×5 mL). The combined filtrates were placed under a nitrogenstream overnight.

[0148] 1.648 g of material was recovered.

[0149] LC-MS: rt=2.23 min, 75%, M+H=334

[0150] 1k: Alternate preparations of Compounds 12,13,14,15,16

[0151] Compounds 12, 13, 14, 15, and 16 are synthesized by the sameprocedure described to make compound 7 using the appropriate startingmaterials.

EXAMPLE 2

[0152] 2A: Insulin—Oral Delivery

[0153] Oral dosing (PO) compositions of delivery agent compound andhuman zinc insulin (minimum 26 IU/mg available fromCalbiochem—Novabiochem Corp, La Jolla, Calif.) were prepared indeionized water. Typically, 500 mg of delivery agent compound was addedto 1.5 ml of water. The solution was vortexed, then heated (about 37°C.) and sonicated. The pH was adjusted to about 7 to 8.5 with NaOH orHCl. Additional NaOH was added, if necessary, to achieve uniformsolubility, and the pH readjusted to about 7 to 8.5. Water was thenadded to bring the total volume to about 2.4 ml and vortexed. About 1.25mg insulin from an insulin stock solution (15 mg/ml made from 0.5409 ginsulin and 18 ml deionized water, adjusting with HCl and NaOH to pH8.15 and to obtain a clear solution using 40 ml concentrated HCl, 25 ml10N NaOH and 50 ml 1N NaOH) was added to the solution and mixed byinverting. The solution may be used in the dosing protocol immediately,or alternatively, the solution may be placed into a 37° C. water bathfor one hour prior to dosing. The final delivery agent compound dose,insulin dose and dose volume amounts are listed below in Table 1.

[0154] The typical dosing and sampling protocols were as follows. MaleSprague-Dawley rats weighing between about 200250 g were fasted for 24hours and administered ketamine (44 mg/kg) and chlorpromazine (1.5mg/kg) 15 minutes prior to dosing and again as needed to maintainanesthesia. A dosing group of five animals was administered one of thedosing solutions. For oral dosing, an 11 cm Rusch 8 French catheter wasadapted to a 1 ml syringe with a pipette tip. The syringe was filledwith dosing solution by drawing the solution through the catheter, whichwas then wiped dry. The catheter was placed down the esophagus leaving 1cm of tubing past the incisors. The dosing solution was administered bypressing the syringe plunger.

[0155] Blood samples were collected serially from the tail artery,typically at time=15, 30, 60, 120 and 180 minutes. Serum insulin levelswere determined with an Insulin ELISA Test Kit (Kit # DSL-10-1600 fromDiagnostic Systems Laboratories, Inc., Webster, Tex.), modifying thestandard protocol in order to optimize the sensitivity and linear rangeof the standard curve for the volumes and concentrations of the samplesused in the present protocol. Serum human insulin concentrations (μU/ml)were measured for each time point for each of the five animals in eachdosing group. The five values for each time point were averaged and theresults plotted as serum insulin concentration versus time. (Previousexperiments revealed no measurable levels of human insulin followingoral dosing with human insulin alone.) The maximum (peak) and the areaunder the curve (AUC) are reported below in Table 1. TABLE 1 Insulin -Oral Delivery Delivery Agent Delivery Compound Insulin Volume Mean PeakAgent Dose Dose dose Serum Compound (mg/kg) (mg/kg) (ml/kg) [INS] ± SD15 200 0.25 1  60.42 ± 117.16 16 200 0.25 1  61.27 ± 116.59 12 200 0.251  83.4 ± 14.69 13 200 0.25 1 76.8 ± 6.80

[0156] 2B: Biotinylated Ribonuclease A (bRNase A) Oral DeliverY

[0157] Oral gavage (PO) dosing solutions of delivery agent compound andbRNase A (Sigma (Milwaukee, Wis.): Ribonuclease A Type XII-A from bovinepancreas) in deionized water were prepared by mixing. The delivery agentcompound solution was prepared in phosphate buffer and stirred. Ifnecessary, the pH of the mixture was adjusted upwards by the addition ofaliquots of NaOH of an appropriate normality until the delivery agentcompound was completely dissolved. The final pH of the dissolveddelivery agent compound was between 7.5 and 9.5. The final dosingsolutions were prepared by mixing 9 volumes of the delivery agentcompound solution with 1 volume of a bRNase A stock solution (20 mgbRNase A in phosphate buffered saline (PBS)). Final concentrations were150 mg/ml delivery agent compound and 2 mg/ml bRNase A.

[0158] The dosing and sampling protocols were as follows. MaleSprague-Dawley rats weighing 200-250 g were fasted for 24 hours andadministered ketamine (44 mg/kg) and chlorpromazine (1.5 mg/kg) 15minutes prior to dosing and again as needed to maintain anesthesia. Adosing group of five animals was administered one of the dosingsolutions in the following manner. An 11 cm Rusch 8 French catheter wasadapted to a 1 ml syringe with a pipette tip. The syringe was filledwith dosing solution by drawing the solution through the catheter, whichwas then wiped dry. The catheter was placed down the esophagus leaving 1cm of tubing past the incisors. The dosing solution was administered bypressing the syringe plunger. Blood samples were collected serially fromthe tail artery at 15, 30, 45, 60 and 90 minutes. Serum bRNase Aconcentrations were quantified by a modified immunoassay as describedbelow.

[0159] Biotinylation of Ribonuclease A

[0160] To label each of the RNase A molecules with one biotin molecule,the ratio of the activated biotin was maintained at 3 moles biotin/1mole RNase A. In a representative biotinylation reaction 500 mg of RNaseA was dissolved in 20 ml of 50 mM NaHCO₃, pH 7.6. 57.08 mg of EZ-LinkSulfo-NHS-LCLC Biotin (Pierce Chemical Company, Rockford, Ill.) wasadded to the solution, dissolved and allowed to stand on ice for 2hours. The reaction mix was then dialyzed (10,000 MW cutoff dialysismembrane (Pierce, Rockford, Ill.)) against 4 liters of PBS at 40 C.overnight. The reaction mix was place in 4 liters of fresh PBS anddialyzed for an additional 4 hours. The dialyzed bRNase A was removedfrom the dialysis membrane, diluted to a final volume of 25 ml with PBS(final concentration of bRNase A=20 mg/ml), and stored a 40 C.

[0161] Assay of Serum Levels of Orally Administered bRNase A

[0162] In general 100 μl aliquots of the rat sera collected at thevarious time points were placed in the appropriate wells of a 96 wellReacti-Bind Streptavidin Coated Polystyrene Plates (Pierce). After a 2hour incubation period the plates were washed and then incubated with apolyclonal rabbit anti-RNase A (Chemicon, Pittsburgh, Pa.). Afterwashing, the plates were incubated for 2 hours with a polyclonal goatanti-rabbit IgG (Chemicon, Pittsburgh, Pa.) conjugated to alkalinephosphatase. The plates were washed after the incubation and the amountof initially captured bRNase A is detected by the addition ofpara-nitrophenyl phosphate (a substrate for alkalinephosphatase)(Pierce, Rockford, Ill.). The amount of bRNase A circulatingin the original rat sera is quantitated by comparison with a standardcurve of bRNAse A which extends from 1000-0.1 ng/mL in fifteen two-folddilutions. The maximum±standard deviation is given in Table 2 below.TABLE 2 Oral Delivery of RNAase Delivery Agent Delivery Compound bRNAaseVolume Agent Dose Dose dose Mean Peak Compound (mg/kg) (mg/kg) (ml/kg)Serum ng/ml 1 150 1 1 2.38 ± 2.2  3 150 1 1 2.98 ± 1.66

[0163] 2c: Oral Delivery of BIBN4096BS

[0164] Oral gavage (PO) dosing solutions of delivery agent compound andthe Calcitonin gene-related peptide antagonist, 1-Piperidinecarboxamide.N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl)carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4(1,4-dihydro-2-oxo-3(2H0-quinazolinyl)-.[R—(R*,S*)]—(BIBN4096BS)in water were prepared. Typically, a solution of the delivery agentcompound was prepared in water and stirred. The final dosing solutionswere prepared by mixing the delivery agent compound with a BIBN4096BSstock solution and diluting to the desired volume (usually 1.0 mL). Ifnecessary, the pH of the mixture was adjusted by the addition ofaliquots of aqueous hydrochloric acid solution of an appropriatenormality until the final pH of the dosing solution was below 7.0. Thefinal compound amounts per dose were 25 mg/kg of BIBN4096BS, 200 mg/kgof delivery agent compound, in a total volume of 1 mL/kg.

[0165] The typical dosing and sampling protocols were as follows. MaleSprague-Dawley rats weighing between 200-250 g were fasted for 24 hoursand administered ketamine (44 mg/kg) and chlorpromazine (1.5 mg/kg) 15minutes prior to dosing. A dosing group of five rats was administeredone of the dosing solutions. For oral gavage (PO) dosing, an 11 cm Rusch8 French catheter was adapted to a 1 mL syringe with a pipette tip. Thesyringe was filled with dosing solution by drawing the solution throughthe catheter, which was then wiped dry. The catheter was placed down theesophagus leaving 1 cm of tubing past the incisors. Solution wasadministered by pressing the syringe plunger. Blood samples werecollected serially from the tail artery, typically at time=0, 15, 30,45, and 60 minutes for oral. Plasma BIBN4096BS concentrations werequantified by using a liquid chromatography/mass spectrometry/massspectrometry assay method using UV detection. The standard range for theassay was 5-2,000 ng/mL. Previous studies indicated baseline values ofabout 10 ng/mL. The maximum is reported below in Table 3. TABLE 3 OralBIBN4096BS Delivery Delivery Agent Delivery Compound BIBN4096BS VolumeAgent Dose Dose dose Mean Peak Compound (mg/kg) (mg/kg) (ml/kg) Serumng/ml  1 200 25 1 28 ± 19  4 200 25 1 23 ± 14  2 200 25 1  453 ± 300*  5200 25 1  402 ± 608* 13 200 25 1  12 ± 3.6 15 200 25 1 0 16 200 25 1  15± 8.1

[0166] The above mentioned patents, applications, test methods, andpublications are hereby incorporated by reference in their entirety.

[0167] Many variations of the present invention will suggest themselvesto those skilled in the art in light of the above detailed description.All such obvious variations are within the fully intended scope of theappended claims.

What is claimed is:
 1. A compound selected from the group consisting ofcompounds 1-18:


2. A composition comprising: (A) an active agent; and (B) at least onecompound of claim
 1. 3. The composition of claim 2, wherein the activeagent is selected from the group consisting of a biologically activeagent, a chemically active agent, and a combination thereof.
 4. Thecomposition of claim 3, wherein the biologically active agent comprisesat least one protein, polypeptide, peptide, hormome, polysaccharide,mucopolysaccharide, carbohydrate, or lipid.
 5. The composition of claim3, wherein the biologically active agent is selected from the groupconsisting of: BIBN-4096BS, growth hormones, human growth hormonesrecombinant human growth hormones (rhGH), bovine growth hormones,porcine growth hormones, growth hormone releasing hormones, growthhormone releasing factor, interferons, α-interferon, β-interferon,γ-interferon, interleukin-1, interleukin-2, insulin, porcine insulin,bovine insulin, human insulin, human recombinant insulin, insulin-likegrowth factor (IGF), IGF-1, heparin, unfractionated heparin,heparinoids, dermatans, chondroitins, low molecular weight heparin, verylow molecular weight heparin, ultra low molecular weight heparin,calcitonin, salmon calcitonin, eel calcitonin, human calcitonin;erythropoietin (EPO), atrial naturetic factor, antigens, monoclonalantibodies, somatostatin, protease inhibitors, adrenocorticotropin,gonadotropin releasing hormone, oxytocin,leutinizing-hormonereleasing-hormone, follicle stimulating hormone,glucocerebrosidase, thrombopoeitin, filgrastim, postaglandins,cyclosporin, vasopressin, cromolyn sodium, sodium chromoglycate,disodium chromoglycate, vancomycin, desferrioxamine (DFO), parathyroidhormone (PTH), fragments of PTH, antimicrobials, anti-fungal agents,vitamins; analogs, fragments, mimetics and polyethylene glycol(PEG)-modified derivatives of these compounds; and any combinationthereof.
 6. The composition of claim 3, wherein the biologically activeagent comprises insulin, BIBN-4096BS, calcitonin, parathyroid hormone,erythropoietin, growth hormones or combinations thereof.
 7. Thecomposition of claim 3, wherein the biologically active agent comprisesBIBN-4096BS.
 8. The composition of claim 3, wherein the biologicallyactive agent comprises insulin.
 9. A dosage unit form comprising: (A)the composition of claim 2; and (B) (a) an excipient (b) a dilutent (c)a disintegrant, (d) a lubricant, (e) a plasticizer, (f) a colorant, (g)a dosing vehicle, or (h) any combination thereof.
 10. The dosage unitform of claim 9, wherein the active agent is selected from the groupconsisting of a biologically active agent, a chemically active agent,and a combination thereof.
 11. The dosage unit form of claim 10, whereinthe biologically active agent comprises at least one protein,polypeptide, peptide, hormone, polysaccharide, mucopolysaccharide,carbohydrate, or lipid.
 12. The dosage unit form of claim 10, whereinthe biologically active agent is selected from the group consisting of:BIBN-4096BS, growth hormones, human growth hormones (hGH), recombinanthuman growth hormones (rhGH), bovine growth hormones, porcine growthhormones, growth hormone releasing hormones, growth hormone releasingfactor, interferons, a-interferon, β-interferon, γ-interferon,interleukin-1, interleukin-2, insulin, porcine insulin, bovine insulin,human insulin, human recombinant insulin, insulin-like growth factor,insulin-like growth factor-1, heparin, unfractionated heparin,heparinoids, dermatans, chondroitins, low molecular weight heparin, verylow molecular weight heparin, ultra low molecular weight heparin,calcitonin, salmon calcitonin, eel calcitonin, human calcitonin;erythropoietin, atrial naturetic factor, antigens, monoclonalantibodies, somatostatin, protease inhibitors, adrenocorticotropin,gonadotropin releasing hormone, oxytocin,leutinizing-hormone-releasing-hormone, follicle stimulating hormone,glucocerebrosidase, thrombopoeitin, filgrastim, postaglandins,cyclosporin, vasopressin, cromolyn sodium, sodium chromoglycate,disodium chromoglycate, vancomycin, desferrioxamine, parathyroidhormone, fragments of PTH, antimicrobials, anti-fungal agents, vitamins;analogs, fragments, mimetics and polyethylene glycol-modifiedderivatives of these compounds; and any combination thereof.
 13. Thedosage unit form of claim 10, wherein the biologically active agentcomprises insulin, BIBN-4096BS, calcitonin, parathyroid hormone,erythropoietin, human growth hormones or combinations thereof.
 14. Thedosage unit form of claim 9, wherein the active agent comprisesrecombinant BIBN-4096BS.
 15. The dosage unit form of claim 9, whereinthe active agent comprises insulin.
 16. The dosage unit form of claim 9,wherein the dosage unit form comprises a dosing vehicle comprising atablet, a capsule, a powder, or a liquid.
 17. The dosage unit form ofclaim 9, wherein the dosing vehicle is liquid selected from the groupconsisting or water, 1,2-propane diol, ethanol, and any combination 18.A method for administering a biologically-active agent to an animal inneed of the agent, the method comprising administering orally to theanimal the composition of claim
 3. 19. A method for preparing acomposition comprising mixing: (A) at least one active agent; (B) thecompound of claim 1; and (C) optionally, a dosing vehicle.